A 

-.  =  Issued  September  17, 1912. 

?=  U.  S.  DEPARTMENT.  OF  AGRICULTURE, 

0  ==-  BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  154. 

8          ~: 

3  . A.  D.  MELVIN,  CHIEF  OP  BUWAU. 

"> 

8  =  *-  //'.    -    •".^.^-^^ 

3  E  E 

2  ~ 

METHODS   OF  CLASSIFYING  THE 
LACTIC-ACID   BACTERIA. 


BY 


LORE  A.  ROGERS, 

Bacteriologist,  Dairy  Division , 
AND 

BROOKE  ].  DAVIS, 
Assistant,  Dairy  Division. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 
1912. 


Issued  September  17, 1912. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  154 

A.  D.  MELVIN,  CHIEF  OF  BUREAU. 


METHODS   OF  CLASSIFYING   THE 
LACTIC-ACID    BACTERIA. 


BY 

LORE  A.  ROGERS, 
Bacteriologist^  Dairy  Division^ 

AND 

BROOKE  J.  DAVIS, 

Assistant,  Dairy  Division. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 
1912. 


Chief:  A.  D.  MELVIN. 

Assistant  Chief:  A.  M.  FARRINGTOX. 

Chief  Clerk:  CHARLES  C.  CARROLL. 

Animal  Husbandry  Division:  GEORGE  M.  ROMMEL,  chief. 

Biochemic  Division:  M.  DORSET,  chief. 

Dairy  Division:  B.  H.  BAWL,  chief. 

Field  Inspection  Division:  R.  A.  RAMSAY,  chief. 

Meat  Inspection  Division:  RICE  P.  STEDDOM,  chief. 

Pathological  Division:  JOHN  R.  MOHLER,  chief. 

Quarantine  Division:  RICHARD  W.  HICKMAN,  chief. 

Zoological  Division:  B.  H.  RANSOM,  chief. 

Experiment  Station:  E.  C.  SCHROEDER,  superintendent. 

Editor:  JAMES  M.  PICKENS. 

DAIRY  DIVISION. 

B.  H.  RAWL,  Chief. 

HELMER  RABILD,  in  charge  of  Dairy  Farming  Investigations. 

S.  C.  THOMPSON,  in  charge  of  Dairy  Manufacturing  Investigations. 

L.  A.  ROGERS,  in  charge  of  Research  Laboratories. 

ERNEST  KELLY,  in  charge  of  Market  Milk  Investigations. 

ROBERT  McADAM,  in  charge  of  Renovated  Butter  Inspection. 

2      - 


LETTER  OF  TRANSMITTAL. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY, 

Washington,  D.  0.,  March  22,  1912. 

SIR:  I  have  the  honor  to  transmit  for  publication  as  a  bulletin  of 
this  bureau  the  accompanying  manuscript  entitled  "Methods  of 
Classifying  the  Lactic-Acid  Bacteria,"  by  Messrs.  Lore  A.  Kogers  and 
Brooke  J.  Davis,  of  the  Dairy  Division  of  this  bureau. 

There  has  hitherto  been  felt  a  need  by  dairy  bacteriologists  and 
others  of  a  classification  of  the  lactic-acid  bacteria  into  naturally 
related  groups  by  means  of  characters  that  can  be  determined  with 
reasonable  accuracy  and  in  a  manner  ordinarily  available.  This 
paper  describes  the  study  of  about  150  cultures  isolated  from  milk, 
butter,  and  cheese,  derived  from  various  parts  of  the  country,  with 
the  object  of  laying  the  basis  for  a  satisfactory  classification. 
Respectfully, 

A.  D.  MELVIN, 

Chief  of  Bureau. 
Hon.  JAMES  WILSON, 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Page. 

Introduction 7 

The  significant  characters  of  the  lactic-acid  bacteria 10 

Morphology 13 

Growth  on  solid  media 14 

Growth  in  milk 15 

Growth  in  broth 17 

Reduction  of  nitrates 17 

Reduction  of  neutral  red 18 

Liquefaction  of  gelatin 18 

Fermentation  of  carbohydrates 19 

Conclusions 28 

References  to  literature ...  29 


ILLUSTRATIONS. 


Page. 
FIG.  1.  Rate  of  acid  formation  in  milk  at  30°  C.  by  cultures  freshly  isolated 

from  milk 14 

2.  Rate  of  acid  formation  in  milk  at  30°  0.  after  26  generations  (2  years) 

on  lactose-agar 15 

3.  Frequency  curve  for  gelatin  liquefaction 19 

4.  Frequency  curves  for  acid  formation  by  the  liquefying  cultures 25 

5.  Frequency  curves  for  acid  formation  by  the  nonliquefying  cultures. . .  26 

6.  Grouping  of  cultures  and  distribution  of  characters  in  groups 27 

5 


METHODS  OF  CLASSIFYING  THE  LACTIC- ACID  BACTERIA. 


INTRODUCTION. 

/ 

The  grouping  of  bacteria  according  to  their  action  on  any  one 
specific  substance  usually  brings  together  bacteria  related  in  that  one 
characteristic  only  but  entirely  unrelated  in  other  respects.  It  is, 
however,  sometimes  convenient  from  a  technical  standpoint  to  group 
bacteria  in  this  way.  The  bacteria  concerned  in  the  souring  of  milk 
have  been  so  grouped  for  so  long  that  many  people  have  come  to 
consider  them  as  a  division  by  themselves  and  their  relation  to  other 
bacteria  has  been  little  considered.  The  bacteria  taking  part  in  the 
souring  of  milk  may  be  readily  divided  into  four  general  groups. 

Group  I  includes  those  bacteria  which  sour  milk  without  peptoniza- 
tion  or  gas  formation ;  they  grow  poorly  on  artificial  media  and  fail  to 
liquefy  gelatin.  Morphologically  they  show  some  variation,  usually 
appearing  as  a  coccus  or  very  short  bacillus  in  pairs  or  in  chains  of 
varying  lengths.  The  bacteria  of  this  group  are  the  ones  ordinarily 
designated  as  the  lactic-acid  bacteria  and  have  been  described  under 
various  names.  They  have  a  very  general  distribution  and  their 
presence  in  milk  is  so  constant  that  they  may  be  considered  as 
normal  inhabitants  of  this  medium. 

Group  II  includes  the  bacteria  forming  an  acid  curd  with  evolution 
of  gas.  This  embraces  varieties  of  BaciUus  coli  and  Bacterium  aerogenes 
or  the  Bacillus  acidi  lactici  of  Heuppe.  The  members  of  this  group 
are  readily  distinguished  from  those  of  Group  I  by  their  abundant 
growth  on  artificial  media,  the  vigorous  evolution  of  gas,  and  the 
marked  difference  in  their  morphology.  An  examination  of  milk 
usually  reveals  their  presence  in  small  numbers,  but  their  number  is 
increased  by  the  influence  of  high  temperatures  or  insanitary  con- 
ditions under  which  the  milk  has  been  collected  or  held. 

Group  III  includes  those  bacteria  forming  an  acid  curd  which  is 
subsequently  partially  peptonized.  The  bacteria  of  this  group  have 
been  little  studied  in  their  relation  to  milk.  It  will  be  shown  that 
this  description  applies  to  varieties  only  distantly  related  to  our 
Group  I  as  well  as  to  some  closely  connected  with  this  type. 

Group  IV  includes  the  high-acid-forming  bacteria  of  which  the 
Bacillus  bulgaricus  is  the  type.  This  organism  is  distinguishable  from 

7 


8  CLASSIFYING  LACTIC-ACID  BACTERIA. 

those  of  the  preceding  groups  by  its  slender  rodlike  form,  its  charac- 
teristic colonies  on  agar,  its  inability  to  grow  in  ordinary  artificial  media> 
and  its  growth  in  the  presence  of  free  acid.  Bacillus  bulgaricus  has 
been  studied  in  its  relation  to  the  fermented  milks  extensively  used  in 
Turkey  and  the  neighboring  countries.  It  has  recently  been 
shown  °  *, 2, 3,  that  it  is  very  widely  distributed  and  may  be  isolated 
from  almost  any  sample  of  mixed  milk.  Its  growth  at  normal 
temperatures  is  so  slow  that  it  is  improbable  that  it  is  a  factor  in  the 
ordinary  souring  of  milk. 

It  is  obvious  that  these  groups  are  connected  only  by  their  ability 
to  ferment  lactose  to  acid  and  the  consequent  precipitation  of  th<> 
casein.  Groups  I,  II,  and  IV  are  evidently  related  to  each  other  in  no 
other  way.  The  identity  of  the  bacteria  of  Group  III  in  so  far  as  they 
occur  as  milk  bacteria  has  not  been  established.  The  need  of  work 
on  methods  of  classification  rather  than  on  descriptions  of  new 
varieties  or  rearrangement  of  old  names  and  descriptions  is  ex- 
emplified by  the  confused  nomenclature  of  the  bacteria  included 
under  Group  I.  We  find  in  the  literature  such  names  as  Bacterium 
lactis  acidi,  Bacillus  lactis  acidi,  B.  acidi  lactici,  B.  guntheri,  and 
Streptococcus  lacticus,  all  of  which,  so  far  as  can  be  determined  by 
published  descriptions,  may  be  included  in  Group  I.  These  names 
are  based  on  variations  in  morphology,  differences  in  growth  on 
artificial  media,  on  the  rate  of  acid  formation  in  milk,  and  various  other 
characteristics  of  doubtful  significance  and  uncertain  stability.  In 
the  light  of  recent  investigations  these  names  and  their  accompanying 
descriptions  have  little  more  than  a  historic  interest.  They  represent 
the  attempt  to  establish  types  by  the  study  of  an  isolated  individual 
organism  with  little  regard  to  its  relation  to  other  similar  individual 
organisms. 

Approached  from  the  standpoint  of  the  dairy  bacteriologists,  the 
lactic-acid  bacteria  have  been  considered  as  a  sharply  defined  group 
peculiar  to  milk.  The  students  of  pathogenic  bacteria  have  boon 
inclined  to  look  on  them  as  a  variety  of  some  of  the  pus-forming 
streptococci. 

The  opinion  that  the  term  lactic-acid  bacteria  covers  a  group  of 
species  or  varieties  and  that  the  names  and  descriptions  already  pub- 
lished do  not  represent  the  true  grouping  is  reflected  in  the  frequent 
attempts  to  establish  means  of  separating  the  group  into  stable 
species  or  varieties.  McDonnell4  attempted  to  do  this,  basing  his 
descriptions  largely  on  the  effect  on  milk.  A  somewhat  similar  course 
was  followed  by  Weigmann.5  Muller8  found  a  correlation  in  the 
solution  of  red  blood  corpuscles  and  the  agglutination  of  immune  sera 
by  the  streptococci  of  sour  milk  and  certain  pathogenic  streptococci, 

a  The  reference  figures  relate  to  the  list  of  references  to  literature  at.  the  end  of  this  bulletin. 


INTRODUCTION.  9 

and  considered  that  this  indicated  a  relationship.  Lohnis7  has  made 
a  classification  of  the  lactic-acid  bacteria  based  largely  on  gas  forma- 
tion, coagulation  of  milk,  formation  of  slime,  and  liquefaction  of 
gelatin.  Conn,  Esten,  and  Stocking8  have  used  in  their  descriptions 
the  action  on  milk  and  the  usual  culture  characteristics.  Heinemann9 
as  a  result  of  his  studies  on  the  bacteria  of  sour  milk,  excludes  the 
name  of  Bacillus  acidi  lactici,  and  concludes  that  aside  from  the  part 
taken  by  B.  aerogenes  and  possibly  by  B.  coli  the  spontaneous  souring 
of  milk  is  brought  about  by  the  Streptococcus  lacticus  of  Kruse,  an 
organism  identical  with  the  common  streptococci  of  sewage  and 
pathological  conditions,  lie  bases  this  conclusion  on  the  similarity 
in  morphological,  cultural,  and  pathogenic  properties. 

All  of  this  work  and  much  more  of  a  similar  nature  adds  little  to  the 
systematic  arrangement  of  the  lactic-acid  bacteria.  It  is  generally  ad- 
mitted that  it  is  difficult  to  identify  cultures  of  any  but'  the  best  known 
and  most  carefully  studied  bacteria  by  the  published  descriptions. 
Cultures  which  seem  identical  when  written  descriptions  are  compared 
are  found  to  be  distinct  when  they  are  grown  side  by  side  under 
uniform  conditions.  This  error  in  identification  is  due  partly  to  the 
difficulty  of  conveying  the  appearance  of  an  object  by  words,  but  in  a 
larger  degree  to  the  instability  and  unessential  nature  of  some  of  the 
characters  employed  in  separating  one  bacterial  species  or  variety 
from  another.  The  inadequacy  of  the  ordinary  methods  is  partic- 
ularly felt  when  one  attempts  a  description  of  the  lactic-acid  bac- 
teria. The  cells  are  small  and  the  morphological  differences  are 
uncertain  and  inconstant.  The  growth  on  solid  media  is  scanty  and 
devoid  of  distinguishing  characteristics.  While  many  of  the  phvsio- 
logical  tests  which  are  found  of  great  value  in  some  groups  fail  when 
applied  to  the  lactic-acid  bacteria,  others,  notably  the  acid  fermenta- 
tions of  sugars,  offer  a  possible  means  of  differentiation  for  members 
of  this  group. 

Variations  in  the  ability  to  ferment  sugars  have  been  observed,  but 
the  use  of  these  variations  in  classifying  or  identifying  cultures  has 
been  limited  for  two  reasons.  There  has  been  a  belief  that  the 
fermentative  power  was  not  constant;  that  this  property  could  be 
lost  or  acquired  so  readily  that  it  could  not  be  used  to  differentiate 
one  culture  from  another  with  any  certainty.  The  more  common 
objection,  however,  is  that  the  separation  on  the  basis  of  sugar  fer- 
mentation divides  the  lactic-acid  bacteria  and  others  possessing  the 
same  general  characteristics,  not  into  natural  groups,  but  into 
innumerable  varieties. 

The  use  of  tests  of  this  kind  in  the  usual  way  by  which  the  fermen- 
tation of,  or  failure  to  ferment,  a  certain  substance  sets  the  culture 
so  reacting  apart  from  all  other  cultures  gives  an  endless  dichotomy 
51194° -12 2 


10  CLASSIFYING   LACTIC-ACID  BACTERIA. 

limited  only  by  the  number  of  test  substances.  Consequently,  the 
ordinary  use  of  sugars  has  increased  rather  than  diminished  the  con- 
fusion now  existing  in  the  classification  of  the  zymogenic  bacteria. 
It  is  obvious  that  what  is  required  in  systematic  bacteriology  is  not 
descriptions  of  new  species  or  a  rearrangement  of  names,  but  the 
establishment  of  means  of  classification  applicable  technically  and 
correct  biologically.  No  one  basis  of  classification  can  be  used  for  all 
groups  of  bacteria,  but  certain  fundamental  principles  should  govern 
any  method  of  arrangement.  Two  of  the  most  obvious  principles  on 
which  the  selection  of  characters  for  classification  should  be  based 
may  be  stated  as  follows:  The  characters  should  be  constant;  they 
should  be  so  selected  that  they  show  real  biological  relationships.  In 
other  words,  bacteria  should  be  arranged  by  means  of  characters  that 
can  be  determined  with  reasonable  accuracy  and  by  means  ordi- 
narily available  into  groups  whose  members  are  related  naturally 
rather  than  by  artificial  bonds,  and  these  characters  should  bo  s<> 
constant  and  so  distinctive  that  identical  organisms  can  always  be 
placed  in  the  same  group. 

This  paper  records  an  attempt  to  determine  which  of  the  charac- 
ters exhibited  by  the  lactic-acid  bacteria  fulfill  these  conditions.  No 
attempt  has  been  made  to  classify  or  name  any  members  of  this  group 
or  to  fix  its  place  in  the  general  bacteriological  system. 

THE  SIGNIFICANT  CHARACTERS  OF  THE  LACTIC-ACID  BACTERIA. 

The  morphology,  staining  reactions,  cell  grouping,  cultural  charac- 
ters, and  growth  in  milk  were  considered,  but  more  attention  was  given 
to  the  fermentation  tests.  We  studied  about  150  cultures  isolated 
from  milk,  butter,  and  cheese  obtained  from  various  parts  of  the 
country.  This  collection  included,  in  addition  to  the  typical  milk- 
curdling,  nonliquefying,  lactic-acid  bacteria,  a  number  of  cultures 
curdling  milk  with  subsequent  digestion  and  which  formed  on  gelatin 
plates  small  saucer-shaped  liquefactions  surrounding  a  solid  colony. 

We  have  determined  on  these  cultures  the  morphology,  Gram's 
stain,  cell  grouping,  in  many  cases  formation  of  capsule,  the  nature 
and  amount  of  growth  on  lactose-agar  slopes  and  in  gelatin  stabs, 
the  rate  of  liquefaction  of  gelatin,  the  nature  of  growth  in  broth, 
growth  in  milk,  the  reduction  of  nitrates  and  of  neutral  red,  and  the 
formation  of  acid  in  broth  containing  various  test  substances.  In 
these  fermentation  tests  we  have  used  the  sugars  lactose,  dextrose, 
galactose,  saccharose,  and  raifinose,  the  alcohols  mannite  and  glyc- 
erin, and  the  polysaccharid  inulin.  The  results  of  these  determina- 
tions are  given  in  Table  1. 


SIGNIFICANT   CHARACTERISTICS. 


11 


TABLE  1. — Significant  characteristics  of  acid-forming  bacteria  derived  from  milk,  butter, 

and  cheese. 


Culture. 

Agar. 

§ 

Gram  stain. 

Cloudiness  in  broth. 

Reduction  of  neutral 
red. 

+  +  +  +  +  1  Curdling  of  milk. 

Reduction  of  nitrates. 

1  Mm.  gelatin  liquefac- 
ooooooooooooooooooooooooooooooooooo  o  |  t  ion  30  days  at  20°. 

Per  cent  lactic  acid  in  broth  after  7  days  at  30"  C. 

1 

o 

j 

{ 

Lactose. 

o 

0.000 
.540 
.522 
.000 
.477 
.405 
.000 
.000 
.531 
.000 
.450 
.405 
.000 
.000 
.405 
.000 
.000 
.000 
.000 
.000 
.531 
.526 
.594 
.009 
.000 
.000 
.000 
.000 
.009 
.000 
.000 
.000 
.000 
.252 
.000 
.000 
.000 
.423 
.027 

a 

O 

0.000 
.000 
.000 
.000 
.000 
.200 
.000 
.000 
.000 
.000 
.027 
.000 
.000 
.018 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.283 
.018 
.000 
.000 
.000 
.000 
.000 
.009 
.027 
.234 
.072 
.000 
.000 

Mannite. 

i 

o 

Rafflnose. 

d 

J_ 

0.000 
.009 
.018 
.009 
.000 
.000 
.009 
.009 
.009 
.000 
.009 
.009 
.000 
.009 
.009 
.009 
.018 
.000 
.009 
.009 
.000 
.018 
.018 
.000 
.009 
.000 
.009 
.000 
.000 
.009 
.009 
.000 
.009 
.009 
.009 
.009 
.009 
.000 
.009 
.009 
.009 
.036 
.000 
.000 
.018 
.009 
.000 
.000 
.000 
.000 
.000 
.000 
.009 
.018 
.216 
.009 
.000 
.000 
.000 
.027 
.387 
.000 
.000 

"."666 

.009 
.000 
.000 

.01S 
.000 
.018 

6es.  . 

t 

I 

44444+ 

+ 

0.360 
.612 
.351 
.382 
.405 
.288 
.364 
.668 
.558 
.400 
.378 
.693 
.365 
.324 
.274 
.369 
.493 
.423 
.378 
.360 
.360 
.585 
.383 
.306 
.594 
.504 
.288 
.540 
.000 
.522 
.396 
.351 
.567 
.432 
.603 
.594 
.468 
.585 
.630 
.360 

0.319 
.360 
.450 
.423 
.378 
.315 
.316 
.315 
.378 
.432 
.414 
.378 
.414 
.414 
.387 
.396 
.333 
.387 
.378 
.360 
.369 
.405 
.324 
.324 
.378 
.369 
.171 
.306 
.162 
.351 
.360 
.369 
.351 
.378 
.360 
.270 
.306 
.495 
.369 
.281 

0.000 
.000 
.009 
.000 
.000 
.279 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.018 
.252 
.000 
.252 
.225 
.000 
.324 
.000 
.360 
.283 
.000 
.000 
.000 
.000 
.000 
.207 
.000 
.324 
.315 
.018 
.081 

0.198 
.234 
.216 
.324 
.378 
.081 
.288 
.270 
.297 
.342 
.283 
.306 
.315 
.342 
.054 
.342 
.405 
.423 
.397 
.351 
.189 
.3% 
.242 
.324 
.369 
.315 
.108 
.225 
.378 
.216 
.306 
.099 
.252 
.000 
.414 
.153 
.297 
.234 
.234 

0.000 
.009 
.000 
.000 
.000 
.171 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.009 
.000 
.000 
.000 
.009 
.000 
.000 
.000 
.000 
.000 
.000 
.009 
.000 
.000 

"."666" 

.009 
.000 
.000 
.000 
.000 
.000 
.027 
.003 
.030 
.000 
.000 
.000 
.009 
.000 
.018 
.018 
.000 
.018 
.018 
.018 
.000 
.000 
.000 
.009 
.000 
.000 
.000 
.009 
.000 
.000 
.000 
.000 
.045 
.000 
.009 
.000 
.000 
.000 

6ex  

6ez  

6fa... 

4. 

6fb  

+ 

4 

6fl  

6fn  

4 

4 

I 

1  +  1  +  1  +  +  +  +  +  +  +  +  +  +  +  1  1  +  1  +  +  +  +  + 

.... 

j 

6fr  

7b  

4 

7c... 

+ 

4 

7d  

7f  

4 

+ 

+ 
+ 

7g... 

7?.  

i 

i 

7k... 

4- 

7n  

4 

7o  

4- 

7p  

4- 

7q  

7s.  . 

4 

7t  

4. 

7u  

4- 

7w  

4 

7x  

4. 

7y  

4. 

7z... 

4. 

7aa  

+ 

7ab.  .  . 

1 

7ad.  

4 

4 

7ae... 

4 

7af  

4 

+ 

J 

7ag  

+ 

7ah  

7ai  

i 

7ai  

i 

7ak  

4- 

+ 

+ 

7ao  

7ap... 

4 

4 

i 

7aq... 

7ar  

4. 

i 

4. 

.279 
.380 
.423 
.363 
.171 
.279 
.342 
.324 
.189 
.207 
.036 
.342 
.396 

.378 
.000 
.000 
.054 
.009 
.387 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.018 
.009 
.693 
.000 
.027 
.288 
.009 
.225 
.009 
.288 
.000 
.324 
.018 
.099 
.036 
.063 

.000 
.072 
.009 
.000 
.342 
.036 
.009 
.009 
.009 
.009 
.000 
.009 
.000 
.000 
.027 
.054 
.000 
.036 
.000 
.000 
.009 
.000 
.018 
.009 
.153 
.000 
.036 
.018 
.036 
.045 
.000 

.342 
.003 
.009 
.018 
.000 
.045 
.000 
.063 
.000 
.063 

"."666" 

.315 
.000 
.018 
.009 
.018 
.000 
.036 
.018 
.397 
.018 
.009 
.018 
.486 
.018 
.378 
.000 
.009 
.018 
.009 

.216 
.234 
.189 
.243 
.171 
.252 
.360 
.396 
.288 
.306 
.234 
.378 
.396 
.360 
.333 
.288 
.441 
.000 
.306 
.324 
.351 
.414 
.261 
.324 
.333 
.414 
.423 
.243 
.302 
.315 
.306 

7as.  

4. 

+ 

4 
4 

1 

:„. 

- 

0 
0 
0 
0 
0 
0 

"6" 
0 

.702 
.513 
.702 
.288 
.180 
.396 
.396 
.306 
.294 
.306 
.270 
:332 

7ay... 

+ 
4. 

+ 

7ba  

7be  

4. 

7bg... 

4, 

7bi.  .  . 

7bj  

7bn  

7bp  , 

7bq  

7br... 

7bs... 

7bt  

- 

- 

t 

~ 

0 
0 
0 
0 
0 
0 
0 

11 

0 

.306 
.423 
.360 
.549 
.297 
.441 
.378 
.360 
.558 
.351 
.225 
.495 
.603 
.432 
.621 
.261 
.540 
.513 

.297 
.360 
.216 
.193 
.387 
.256 
.144 
.221 
.297 
.144 
.103 
.297 
.248 
.260 
.270 
.297 
.648 
.261 

Tbv  

7bx  

7by... 

4. 

7bz  

4 

+ 

7ca  

I 

7cb  

7cc  

7cd...,  

7ce  

7cf  
7cg  

\ 

4 

4 

4- 

- 

"6" 

0 
0 
0 
0 
0 
0 

7ch... 

7ci  

7ci  

7ck... 

7cl  

7cm... 

4 

12 


CLASSIFYING   LACTIC-ACID  BACTERIA. 


TABLE  1 .— Significant  characteristic*  of  acid-forming  bacteria  derived  from  milk,  butter, 

and  cheese — Continued . 


Agar. 

5 

Gram  stain. 

Cloudiness  in  broth. 

3 

I 

\ 

Curdling  of  milk. 

Reduction  of  nitrates. 

•  • 

Per  cent  lactic  acid  in  broth  after  7  days  at  30*  C. 

Culture. 

9 

j 

Mm.  geiauu  nqi 
t  ion  30  days  at 

Dextrose. 

, 

j 

3 

£ 

O 

1 

1 

7cn  

4- 
4- 
4- 
4- 

4- 
4- 

4- 

4- 
4- 
4- 
4- 

4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 

4- 

4- 
4- 
4- 

4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 

+ 
4- 
4- 
4- 
4- 
4- 
4- 
4- 

4- 
4- 
4- 

4- 

4- 
4- 

4- 
4- 

4- 

H 

4- 

H 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

6 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
9 
14 

378 
351 
576 
576 
459 
522 
.684 
.261 
.351 
.316 
.423 
.495 
.387 
.405 
.316 
.279 
.369 
.333 
.369 
.423 
.225 
.171 
.396 
.405 
.396 
.288 
.216 
.432 
.351 
.288 
.414 
.334 
.378 
.594 
.531 
.378 
.522 
.477 
.486 

"."495 
.468 
.432 
.603 
.495 
.720 
.324 
.234 
.369 
.324 
.220 
.360 
.230 
.211 
.165 
.270 
.203 
.149 
.432 
.099 
.207 
.243 
.250 
.495 
.652 
.216 
.633 
.171 
.180 
.198 
.198 

234 
206 
274 
225 
265 
.243 
.297 

000 
099 
027 
009 
009 

000 
000 
000 
000 
009 

018 
027 
000 
009 
243 
117 
036 
009 
.531 
.549 

477 
342 
441 
450 
522 

000 
009 
225 
000 
000 

0.000 
.018 
.000 
.000 
.009 
.036 
.009 
.018 
.270 
.000 
.000 
.018 
.306 
.000 
.000 
.252 
.216 
.288 
.279 
.288 
.261 
.000 
.306 
.000 
.297 
.342 
.108 
.630 
.306 
.270 
.306 
.000 
.000 
.000 
.027 
.000 
.000 
.000 
.008 
.009 
.000 
.000 
.000 
.000 

.oos 

.008 
.008 

.ooc 

.01$ 
.01$ 
.OK 
.OOC 
.01$ 
.01$ 

.03< 
.01$ 
.00! 
.00! 
.00! 
.00! 
.011 
.001 
.001 
.001 
.001 
.01 
.001 

7cp  

7cr  

(CS  

7ct 

.000 

.009 
.009 
.162 
.198 
.225 
.045 
.117 
.099 
.045 
.198 
.252 
.234 
.261 
.162 
.234 
.198 
.234 
.000 
.027 
.315 
.225 
.198 
.180 
.216 
.252 
.180 
.189 
.000 
.000 
.000 
.000 
.000 
.000 
.018 
.018 
.000 
.000 
.018 
.009 
.000 
.000 
.048 
.207 
.216 
.027 
.162 
.072 
.054 
.081 
.180 
.086 
.207 
.315 
.234 
.054 
.027 
.045 
.036 
.045 
.009 
.045 
.000 
.  121 
.072 
.036 

.405 
.108 
.279 
.216 
.081 
.279 
.234 
.036 
.378 
.252 
.279 
.324 
.252 
.279 
.243 
.261 
.279 
.153 
.288 
.198 
.288 
.558 
.234 
.270 
.288 
.198 
.270 
.288 
.351 
.216 
.351 
.270 
.171 
.315 
.342 
.360 
.143 
.369 
.225 
.036 
.063 
.099 
.239 
.234 
.099 
.175 
.036 
.072 
.144 
.146 
.198 
.203 
.135 
.144 
.058 
.063 
.081 
.256 
.261 
.075 
.279 
.054 
.090 
.081 
.287 

.000 
.000 
.630 
.630 
.648 
.018 
.603 
.054 
.000 
.630 
.648 
.630 
.603 
.567 
.487 
.639 
.648 
.703 
.414 
.676 
.639 
.261 
.676 
.756 
.657 
.630 
.648 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.009 
.000 
.000 
.009 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.027 
.018 
.018 
.000 
.000 
.009 
.045 
.000 
.027 
.000 
.018 
.000 
.000 
.036 
.054 
.018 

.108 
.414 
.441 
.018 
.372 
.423 
.432 
.396 

."423" 
.414 
.441 
.414 
.405 
.405 
.450 
.009 
.432 
.414 
.414 
.423 
.441 
.441 
.414 
.423 
.441 
.432 
.369 
.351 
.405 
.351 
.369 
.387 
.405 
.387 
.333 
.343 
.369 
.045 
.108 
.171 
.216 
.194 
.144 
.270 
.144 
.126 
.144 
.077 
.275 
.176 
.316 
.234 
.171 
.175 
.153 
.324 
.355 
.153 
.324 
.171 
.117 
.121 
.144 

.009 
.531 
.459 
.029 
.009 
.549 
.018 
.459 
.450 
.477 
.522 
.531 
.549 
.468 
.504 
.540 
.018 
.558 
.522 
.540 
.549 
.468 
.369 
.612 
.423 
.414 
.000 
.000 
.000 
.000 
.540 
.000 
.000 
.000 
.000 
.000 
.000 
.072 
.000 
.585 
.027 
.274 
.009 
.014 
.000 
.252 
.153 
.285 
.351 
.351 
.198 
.369 
.297 
.036 
.192 
.045 
.018 
.018 
.036 
.018 
.045 
.045 
.054 
.090 

i 

.504 
.045 
.531 
.018 
.009 
.567 
.558 
.558 
.558 
.549 
.558 
.549 
.595 
.054 
.531 
.549 
.567 
.558 
.540 
.549 
.594 
.540 
.558 
.000 
.000 
.054 
.018 
.000 
.009 
.000 
.000 
.000 
.000 
.000 
.000 
.000 
.369 
.072 
.191 
.406 
.004 
.243 
.004 
.000 
.297 
.135 
.162 
.230 
.216 
.297 
.018 
.045 
.036 
.018 
.009 
.009 
.018 
.000 
.027 
.018 
.027 

7da  

4- 

4- 
4- 
4- 

4- 

4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 

+ 
4- 
4- 
4- 
4- 

7dc  

7dd 

7df 

7de 

7dfi 

7di 

7di 

7dk  

7dl 

7dm 

7dn 

7do 

7dp 

7ds 

7du 

7dv 

7dw 

7dx 

7dv 

7dr 

lOav 

4- 

4- 
4- 
4- 
4- 

4- 

4- 
4- 
4- 

4- 
4- 

4- 

+ 
4- 

4- 

+ 
4- 

4- 

4- 
4- 
4- 

4- 
4- 
4- 
4- 

4- 

4- 

4- 
4- 
4- 
4- 

4- 

lOaw 

4- 
4- 

4- 

lOax 

lObb  

4- 

+ 
4- 

4- 
4- 

4- 

4- 

4- 
4- 
4- 
4- 
4- 
4- 
4- 

lObd  

lObe  
lObg        .     . 

lObn 

lObi  

lObo 

lObr 

4- 

4- 
4- 

4- 

4- 

lObt     

4- 

4- 

4- 
4- 
4- 
4- 

4- 

4- 
4- 
4- 
4- 

+ 
4- 

4- 
4- 
4- 

4- 
4- 

lObl  

4- 
4- 
4- 
4- 
4- 

13c  

4- 

13f 

4- 
4- 

13h  

26 

2' 

21 
6 
10 
18 
13 
15 
25 
15 
1 

i 
1 
l 

l 
i 
13 
1' 

4. 

13m... 

.+. 

4- 

4- 
4- 

13o  
13r  

4- 

13t  

i 

13u 

4- 

+ 

13w 

4- 

13x 

4- 

4- 

13v 

• 

4- 

4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 
4- 

4- 
4- 

4- 

4- 

4- 
4- 
4- 
4- 
4- 
•4- 
4- 

13aa 

4- 

13ab 

4- 

4- 

13ac  
13ad 

4- 

4- 

13as;  
13ah  
13ai       

+ 

4- 

ia  

4- 
4- 

13am     

4- 

13ap  

4- 

MORPHOLOGY. 


TABLE  1. — Significant  characteristics  of  acid-forming  bacteria  derived  from  milk,  butter, 

and  cheese — Continued . 


Ag 

ar. 

.a 

I 

1 
1 

1- 

Per  c 

'lit  lac 

tic  in  -i> 

linbr 

ath  aft 

er  7  da 

ysat  3 

oec. 

e 

§ 

44 

o-~ 

a 

S  « 

Culture. 

c 

"3 

a 

'I 

°l 
ss 

E 
"3 

*S 

S 

a  £ 

a-S 

| 

. 

. 

•p 

W 

a 
•3 

3 

a 

'1 

t£?0 

.  o 

1 

| 

1 

i 

| 

I 

a 

a 

% 

f 

s 

£ 

B-2 

K 

3 

9, 

~ 

"3 

S 

i 

O 

• 

<5 

0 

O 

« 

6 

« 

S 

0 

S 

O 

9 

O 

« 

a 

13ar  

+ 

_ 

0.198 

0.027 

0.027 

0.009 

0.  153 

0.036 

13at  

_1 

1 

18 

0  162 

171 

054 

054 

027 

117 

13av  

+ 

_ 

+ 

_ 

11 

.171 

.145 

.045 

.045 

.000 

.063 

.036 



13aw  

-f 

1 

I 

7 

117 

135 

090 

036 

009 

13ax  

-f. 

, 

5 

126 

181 

'ooo 

027 

000 

090 

13be  

, 

12 

216 

135 

054 

090 

000 

099 

027 

13bg  

• 

, 

13 

189 

136 

036 

072 

009 

054 

072 

13bk 

297 

207 

126 

009 

009 

189 



13bl  

T 

234 

117 

054 

036 

0^7 

108 

081 

MORPHOLOGY. 

The  cultures  examined  showed  four  more  or  less  distinct  types. 
The  liquefying  group  included  a  number  of  cultures  of  micrococci, 
with  frequent  grouping  in  tetrads.  This  was  associated  with  good 
growth  on  agar  and  certain  fermentative  reactions  which  made  them 
easily  distinguishable  from  the  liquefying  cultures  with  the  morphol- 
ogy of  the  typical  lactic  bacteria.  The  nonliquefiers,  excepting  a 
few  cultures  of  micrococci,  showed  three  variations.  Cells  may  be 
nearly  or  quite  round.  When  in  this  condition  they  are  usually 
found  in  chains  of  four  or  more  cells.  Single  cells  or  pairs  of  cells 
are  almost  always  oval  and  sometimes  are  distinctly  of  the  bacillus 
type.  A  slight  variation  is  sometimes  found  in  that  the  cells  are 
somewhat  pointed  at  one  end.  All  of  these  types  can  usually  be  found 
in  the  same  culture  and  not  infrequently  in  the  same  microscopic 
field. 

The  question  of  the  classification  of  the  lactic-acid  bacteria  as  cocci 
or  as  bacilli  has  been  much  debated  and  is  yet  in  an  unsettled  condi- 
tion. The  formation  of  chains,  on  which  much  emphasis  is  placed 
by  some  writers,  is  not  pertinent,  as  the  tendency  to  form  chains  is 
as  common  among  the  bacilli  as  among  the  streptococci.  In  our 
present  state  of  knowledge  the  proper  placing  of  these  bacteria  is 
largely  a  question  of  opinion  or  of  definition.  No  satisfactory  settle- 
ment can  be  reached  until  we  have  sufficient  knowledge  to  establish 
the  natural  relations  of  the  lactic-acid  bacteria  with  other  groups. 
Even  in  this  case  it  is  probable  that  close  relationship  will  be  traced 
on  the  one  hand  with  groups  that  are  distinct  cocci  and  on  the  other 
with  groups  that  are  unquestionably  bacilli. 

It  may  be  stated  in  this  connection  that  one  of  our  cultures  (7dv), 
not  differing  morphologically  from  other  cultures  of  the  typical 


14 


CLASSIFYING   LACTIC-ACID  BACTERIA. 


lactic  type,  was  actively  motile.  This  culture  was  pronounced  by 
Dr.  Heinemann  to  bo  a  typical  Streptococcus  lacticus,  except  that  it 
did  not  curdle  milk  promptly. 

All  of  these  cultures  stain  readily  and  are  Gram  positive.  A  cap- 
sule could  usually  be  demonstrated  if  the  test  was  repeated  under 
varying  conditions,  indicating  that  it  is  formed  only  under  certain 
circumstances.  The  circumstances  under  which  a  capsule  was  found 
indicated  that  it  was  in  some  way  connected  with  the  acidity  of  the 
culture. 


0/234 

FIG.  1.— Rate  of  acid  formation  In  milk  at  30*  C.  by  cultures  freshly  Isolated  from  milk. 

GROWTH   ON    SOLID   MEDIA. 

Little  need  be  said  under  this  he^d,  although  the  growth  of  the 
lactic-acid  bacteria  on  agar  and  gelatin  has  been'described  in  great 
detail.  The  growth  is  always  scanty,  and  the  variations,  which  are 
very  slight,  are  due  more  to  differences  in  the  chemical  and  physical 
condition  of  the  medium  than  to  varietal  distinction. 

Some  real  variation  may  be  observed  in  the  size  of  mature  gelatin 
colonies,  but  this  is  so  influenced  by  the  medium  and  the  number  of 
colonies  on  the  plate  that  it  is  of  little  value.  Variations  of  this  kind 
are  probably  merely  expressions  of  a  tolerance  or  intolerance  to 
certain  conditions  which  could  be  determined  with  much  greater 


GROWTH   IN   MILK. 


15 


accuracy  by,  other  methods.  The  reaction  of  a  particular  lot  of 
gelatin  may  produce  large  colonies  of  one  culture  while  it  limits  the 
growth  of  another  culture  to  colonies  of  almost  microscopic  si/c. 


GROWTH    IN    MILK. 


The  time  required  to  curdle  milk  under  definite  conditions  has  been 
employed  almost  universally  in  describing  lactic-acid  bacteria, 
although  it  is  generally  admitted  that  this  property  is  variable, 
especially  after  the  culture  has  been  grown  under  artificial  conditions. 
This  variation  is  illustrated  by  figures  1  and  2. 


.7 


.5 


.2 


'  *  3  4  S 

FIG.  2.— Rate  of  acid  formation  in  milk  at  30°  C.  after  26  generations  (2  years)  on  actose-agar. 

Figure  1  shows  a  variation  in  the  freshly  isolated  cultures  from  7b, 
which  failed  to  curdle  milk  in  5  days,  to  7k,  which  curdled  milk 
promptly,  forming  nearly  1  per  cent  of  acid  in  48  hours.  Two  years' 
growth  under  uniform  conditions  redxiced  these  differences  materially. 
The  weaker  cultures  changed  little  or  not  at  all,  but  the  more  active 
ones  lost  much  of  their  vigor;  that  is  to  say,  long-continued  growth 
under.uniform  conditions  tended  to  reduce  these  cultures  to  a  common 
level.  It  is  not  easy  to  restore  this  lost  vigor.  Repeated  transfers 
in  milk  increased  the  activity  of  some  of  the  cultures,  but  failed  to 
bring  the  more  active  ones  back  to  the  rapid  fermentation  of  the  fresh 
cultures.  It  is  probable  that  these  differences  are  due  to  a  variation 
in  the  vitality  rather  than  to  a  variation  in  the  particular  function 


16 


CLASSIFYING    LACTIC-ACID  BACTERIA. 


of  forming  acid  from  sugar.  In  these  studies  it  was  frequently 
observed  that  those  cultures  curdling  milk  tardily  or  not  at  a)l  multi- 
plied slowly  and  never  attained  the  numbers  reached  by  the  cultures 
curdling  milk  in  a  short  time.  This  is  illustrated  by  Table  2,  in  which 
is  given  the  acid  formation  and  rate  of  multiplication  of  two  cultures, 
one  curdling  milk  in  a  short  time  and  one  failing  to  curdle  milk  in  '2\ 
hours.  Flasks  of  milk  were  inoculated  from  fresh  milk  cultures  and 
incubated  at  30°  C. 

TABLE  2. — Relative  rate  of  multiplication  and  acid  formation  in  milk. 


Slow  acid  former. 

Kiipidacid  former. 

Hours 

from 

Inocula- 

Bacteria 

Bacteria 

tion. 

Acidity. 

per  cubic 

Acidity. 

per  cubic 

centimeter. 

centimeter. 

Per  cent. 

Per  cent. 

0 

0.216 

357,000 

0.220 

350,000 

3 

.216 

586.000 

.225 

650,000 

6 

.225 

1,690,000 

.225 

2,900,000 

9 

.225 

16,900,000 

.225 

46,000,000 

12 

.230 

59,500,000 

.261 

157,000,000 

24 

.306 

710,000,000 

.900 

1,830,000,000 

It  will  be  observed  that  in  a  general  way  the  acidity  in  each  case 
is  proportioned  to  the  number  of  cells  present.  This  is  in  accordance 
with  the  observation  of  Rahn,10  who  calculated  the  amount  of  acid 
formed  in  relation  to  the  number  of  cells  in  the  culture  and  found 
that  this  ratio  was  constant,  although  when  only  a  small  number  of 
bacteria  were  present  the  amount  of  acid  was  so  small  that  it  could 
not  be  measured  by  ordinary  methods.  Schierbeck,11  who  studied 
this  form  of  variation  in  the  lactic-acid  bacteria,  found  that  by  replat- 
ing  and  making  subcultures  new  cultures  could  be  obtained,  some  of 
which  followed  the  active  fermentation  of  the  original,  while  others 
were  slow  acid  formers.  In  some  of  these  cultures  the  rate  of  acid 
formation  could  not  be  varied  by  subsequent  plating  and  selection, 
but  cultures  in  which  the  ability  to  ferment  lactose  rapidly  seemed 
to  be  fixed  could  be  changed  to  slow  fermenters  by  growing  them  in 
milk  containing  a  small  amount  of  carbolic  acid.  Buchanan  and 
Truax  lf  attempted  to  fix  strains  of  the  lactic-acid  bacteria  by  selec- 
tion and  transfer  from  tubes  of  lactose  broth  showing  wide  differ- 
ences in  acidity.  They  failed  entirely  and  concluded  that  "im- 
pressed variations  do  not  appear  to  be  heritable."  It  seems  propa- 
ble  that  they  were  unable  to  fix  these  variations  because  they  were 
working,  not  with  variations  in  a  function,  but  with  the  rate  of 
multiplication,  something  which  may  be  controlled  by  an  endless 
chain  of  circumstances.  For  the  same  reasons  this  characteristic 
can  not  be  successfully  used  as  a  basis  for  classification. 


REDUCTION    OF    NITRATES.  17 

GROWTH  IN  BROTH. 

The  growth  in  a  medium  of  this  nature  should  be  considered  as  the 
expression  of  certain  peculiarities  not  evident  on  superficial  examina- 
tion. A  cloudiness  or  turbidity  is,  with  the  lactic-acid  bacteria,  an 
evidence  of  rapid  growth,  and  it  is  governed  more  by  external  condi- 
tions than  by  the  nature  of  the  culture.  Cultures  which  remain  clear 
in  nonsaccharine  broth  may  be  cloudy  when  certain  sugars  are  added, 
and  others  may  show  cloudiness  when  dibasic  potassium  phosphate 
is  added,  but  not  in  its  absence.  Profuse  cloudiness  is  usually  fol- 
lowed by  a  clearing  and  the  accumulation  of  a  sediment  coinciding 
with  the  checking  of  the  growth  by  increasing  acidity.  Some  few 
cultures,  however,  never  show  turbidity,  and  the  broth  remains  per- 
fectly clear  with  no  evidences  of  growth,  although  a  high  acidity  is 
produced.  This  is  perhaps  due  to  the  tendency  to  form  tangled 
chains  possessing  in  the  aggregate  a  specific  gravity  greater  than  the 
broth.  Muller  13  states  that  a  clouded  bouillon  is  associated  with  the 
formation  of  single  cells  or  pairs,  while  a  clear  bouillon  is  coordinated 
with  the  formation  of  long  chains.  In  his  work  he  found  that  the 
cultures  that  never  clouded  the  bouillon  were  the  "Gait  Stamme" 
found  associated  with  yellow  mammitis.  When  the  failure  to  cloud 
the  broth  is  fixed  and  constant  it  may  be  of  some  assistance  in  clas- 
sification. 

REDUCTION     OF     NITRATES. 

The  reduction  of  nitrates  to  nitrites  was  determined  by  growth  in 
the  following  medium  for  7  days  at  30°  C. 

Peptone gram. .       1.  0 

Potassium  nitrate gram . .       0.  2 

Water  (distilled) c.  c. .  1, 000 

Of  the  147  cultures  tested  for  the  reduction  of  nitrates,  17  gave  a 
positive  reaction.  All  of  these  were  differentiated  from  the  typical 
lactic-acid  bacteria  by  one  or  more  coordinated  characters.  Twelve 
were  of  the  liquefying  tetrad  group.  Three  (6fl,  7aa,  and  7ak),  were 
gas  formers,  and  7ak  was  in  addition  a  bacillus  larger  than  the  lactic 
type  and  grew  readily  on  solid  media.  One  (7cc)  resembled  the 
lactic  type  in  morphology  but  the  cells  were  larger,  and  in  addition 
to  liquefying  gelatin  and  giving  an  abundant  growth  on  agar  it  made 
milk  slimy.  Culture  7dw  was  a  small  micrococcus. 

None  of  the  cultures  belonging  to  Group  I  gave  any  evidence  of 
growth  in  this  medium.  It  is  therefore  of  no  value  in  making  sub- 
divisions of  this  group,  but  may  be  a  convenient  means  for  the  detec- 
tion of  cultures  resembling  the  type  in  many  features  but  differing 
in  certain  salient  points. 


18 


CLASSIFYING   LACTIC-ACID  BACTERIA. 


REDUCTION  OF  NEUTRAL  RED. 


In  making  this  test  the  following  medium  was  used: 
Broth  (neutral) c.  c. .  1, 000 


Dextrose • grams. 

One-half  per  cent  solution  Griibler's  neutral  red .  .c.  c. 


5 
10 


The  neutral  broth  was  made  as  follows: 

Beef  extract grams. 

Peptone grams. 


4 

10 
Water c.  o..  1,000 

The  tubes  were  examined  after  incubating  at  30°  C.  for  7  days  in 
an  anaerobic  jar  from  which  the  oxygen  was  exhausted  by  absorp- 
tion with  pyrogallic  acid.  Gordon  u  considers  this  test  of  diagnostic 
value.  It  has,  however,  the  disadvantage  of  not  always  giving  definite 
results,  although  with  nearly  all  cultures  the  reduction  was  either 
nil  or  very  evident.  Of  the  36  cultures  reducing  neutral  red,  a  large 
proportion  ferment  the  more  resistant  test  substances,  such  as  sac- 
charose, glycerin,  mannite,  and  raffinose,  and  7,  or  19  per  cent  of  the 
whole,  liquefy  gelatin.  None  of  those  liquefying  gelatin  ferment 
raffinose,  while  of  the  29  nonliquefying  cultures  reducing  neutral  red 
75  per  cent  ferment  raffinose.  There  is  a  correlation  between  the 
reduction  of  neutral  red,  the  liquefaction  of  gelatin,  and  the  fer- 
mentation of  saccharose,  glycerin,  and  mannite  in  one  group  and 
between  neutral  red,  saccharose,  glycerin,  mannite,  raffinose,  and 
inulin  in  another.  These  correlations  are  evident  in  figure  6.  The 
faculty  of  reducing  neutral  red  seems  to  be  usually  coordinated  with 
other  reactions  and  is  therefore  of  some  value  in  differentiating 
cultures. 

LIQUEFACTION   OF    GELATIN. 

The  value  of  this  test  is  too  generally  recognized  to  need  discus- 
sion. In  our  work  we  have  used  Clark  and  Gage's  method  of  reducing 
the  rate  of  liquefaction  to  mathematical  terms,  ignoring  the  appear- 
ance of  the  culture.  The  gelatin  tubes  were  inoculated  by  spreading 
a  few  drops  of  a  fluid  culture  on  the  surface  of  the  medium.  The 
line  of  the  surface  was  marked  on  narrow  strips  of  paper  pasted  on 
opposite  sides  of  the  tube,  and  the  cultures  were  incubated  at  18° 
to  20°  C.  At  the  end  of  30  days  the  amount  of  liquefaction  was 
measured  and  expressed  as  millimeters  of  liquefaction.  The  results 
of  this  test  are  tabulated  in  Table  3. 

TABLE  3. — The  liquefaction  of  gelatin. 


Liquefaction  millimeters.  . 
Nuaiber  of  cultures  

0 

108 

ItoS 
3 

6  to  10 
10 

11  to  15 
10 

'    16  to  20 
2 

21  to  25 
•       3 

Over  25 
2 

Peri3ent  of  total  

78.3, 

2  2 

7  2 

7  2 

1  4 

2  2 

1  4 

FERMENTATION   OF  CARBOHYDRATES.  19 

These  results  are  platted  in  figure  3   to  show  the  frequency  <>f 
occurrence  of  certain  arbitrary  types. 

This  curve  gives  some  indication  of  a  division  on  the  basis  of 
gelatin  liquefaction  into  three  types,  one  failing  to  liquefy,  one 
liquefying  6  to  15  millimeters,  and  one  20  to  25  millimeters.  How- 
ever, the  total  number  of  liquefying  cultures  was  so  small  that  it  is 
safe  to  make  a  division  into  liquefiers  and  nonliquefiers  only,  and  to 
depend  on  other  tests  for  further  division  of  the  liquefiers.  Even 
the  separation  of  the  liquefiers  from  the  nonliquefiers  is  not  entirely 
reliable,  as  it  is  well  known  that  this  character  is  vari- 
able and  under  some  conditions  may  be  entirely  lost. 

If  physiological  tests  are  of  value  they  should  show 
by  correlation  or  lack  of  correlation  which  cultures 
70  \  belong  properly  with  the  nonliquefiers  and  which  are 

members  of  liquefying  varieties  in  which  the  ability  to 
produce  a  proteolytic  enzym  has  been  lost. 

FERMENTATION    OF   CARBOHYDRATES. 

Mention  has  already  been  made  of  the  objections  to 
the  use  of  the  fermentation  of  sugars  and  similar  sub- 
stances. The  question  of  the  constancy  of  these  reac- 
tions has  been  the  subject  of  investigation,  and  while 
there  is  some  disagreement  the  opinion  of  those  who 
have  studied  the  question  most  carefully  seems  to  be 
that  they  are  at  least  as  constant  as  any  of  the 
characters  ordinarily  used  in  classification.  Twort,15 

working  with  gas- 
forming  cultures, 
was  able  to   in- 
—     duce  acid 


/O 


/-S      6-/0     fM5     f6-2O    2J-25 

m.m,  of  //yet e faction.  tlon  fr°m  sugars 

FIG.  3.-Frequency  curve  for  gelatin  liquefaction.  Which  the  organ- 

ism  originally  did 

not  ferment  by  repeated  transfers  in  a  medium  in  which  this  sugar  was 
the  only  carbohydrate  furnished.  Each  transfer  was  held  14  days  to 
allow  the  cells  to  work  on  the  sugar  after  other  sources  of  food  had  been 
exhausted.  Kitchie  16  concluded  that  while  cultures  of  Bacillus  coli 
tested  at  different  times  gave  constant  fermentation  reactions,  the 
streptococci  were  inconstant.  Gordon17  tested  the  constancy  of  11 
cultures  by  passing  them  through  mice.  One  culture  lost  ability  to 
reduce  neutral  red  and  one  gained  ability  to  ferment  salicin.  All 
others  remained  unchanged.  In  all  of  this  work  the  fermentative 
ability  was  determined  by  growing  the  organism  in  broth,  with  the 
addition  of  the  test  substance  and  litmus,  and  the  change  of  the 


20  CLASSIFYING   LACTIC-ACID  BACTERIA. 

litmus  from  blue  was  taken  as  a  positive  reaction.  A  slight  change 
in  the  reaction  of  the  medium  may  change  litmus  from  blue  to  rod, 
and  this  acidity  may  be  formed  from  some  substance  other  than  the 
sugar.  The  reduction  of  the  litmus  is  certainly  not  an  indication  of 
the  fermentation  of  the  test  substance.  The  work  of  MacOonkey  18 
indicates  that  under  natural  conditions  these  reactions  are  constant 
and  of  value  in  differentiation.  Among  the  cultures  examined  \v<-iv 
15  cultures  of  B.  typhosus  varying  from  one  freshly  isolated  to  one 
grown  16  years  on  artificial  media.  These  gave  identical  fermenta- 
tion reactions  when  tested  with  various  carbohydrates. 

In  another  paper  the  same  investigator  states  that  the  fermentative 
reactions  of  B.  coli  remained  unchanged  after  a  long  exposure  to  un- 
favorable conditions.  He  expresses  the  opinion  that  one  group  is 
not  derived  from  another  by  the  loss  of  characters. 

Harding,19  working  with  Pseudomonas  campestris,  an  organism 
pathogenic  to  certain  plants,  obtained  somewhat  similar  results.  Of 
the  four  substances  used  for  fermentation  tests  this  organism  attacked 
only  one,  but  this  and  all  other  physiological  tests  employed  were 
identical  for  the  44  cultures  collected  from  various  parts  of  the 
country. 

In  our  own  work  no  systematic  investigation  was  undertaken  to 
determine  the  constancy  of  the  fermentation  reactions,  but  all  our 
observations  tend  to  prove  that  the- property  of  forming  acid  from 
carbohydrates  and  similar  substances  is  not  easily  lost  or  acquired. 
One  culture  showing  no  evidence  of  ability  to  ferment  saccharose 
was  carried  for  100  generations,  or  a  period  of  about  one  year,  on  a 
saccharose-agar.  At  the  end  of  this  period  the  culture  still  showed 
no  fermentation  of  saccharose  and  the  lactose  fermentation  remained 
unchanged. 

In  no  case  did  any  of  our  cultures  show  any  change  in  fermentative 
ability  on  repeated  tests.  It  not  infrequently  happens  that  a  culture 
failing  entirely  to  give  an  acid  reaction  on  the  first  test  showed  an 
active  fermentation  when  the  test  was  repeated,  but  this  was  evi- 
dently due  to  a  failure  of  the  inoculation  rather  than  to  a  change  in 
the  organism.  Many  of  the  cultures  grew  so  poorly  on  artificial 
media  that  they  were  propagated  with  difficulty  and  transfers  fre- 
quently failed  to  grow.  A  large  proportion  of  the  cultures  were 
subjected  to  these  tests  two  or  three  times,  some  of  them  at  intervals 
of  several  months.  The  second  test  almost  always  agreed  with  the 
first  not  only  in  the  presence  or  absence  of  fermentation  but  also  in 
the  amount  of  acid  formed.  This  is  illustrated  by  Table  4,  which 
contains  results  on  the  fermentation  of  lactose.  These  figures  were 
picked  at  random 


FERMENTATION   OF   CARBOHYDRATES. 
TABLE  4. — Showing  constancy  of  fermentation  of  lactose. 


21 


Test. 

Acidity  of  broth  expressed  as  per  cent  of  lactic  acid. 

Firet  

0.112 
.117 

0.171 
.261 

0.216 
.171 

0.387 
.306 

0.288 
.225 

0.000 
.144 

0.180 
.261 

0.306 
.288 

0.153 
.135 

0.108 
.009 

0.252 
.243 

Second  

With  some  of  the  test  substances  the  reaction  was  always  very 

«/  •/ 

positive;  that  is,  the  reaction  remained  unchanged  or  a  considerable 
acidity  was  developed.  This  was  especially  noticeable  with  sac- 
charose, mannite,  and  raffinose.  With  others,  particularly  with 
glycerin,  the  acid  was  developed  slowly  and  in  such  small  quantities 
that  it  was  sometimes  difficult  to  determine  if  there  was  a  real  fermen- 
tation or  a  slight  change  in  the  reaction  which  was  independent  of 
the  test  substance.  In  doubtful  cases  a  retest  usually  gave  definite 
results. 

It  is  not  to  be  expected  that  a  character  of  this  kind  would  be 
absolutely  fixed.  Indeed,  the  fact  that  one  culture  attacks  a  certain 
sugar  while  similar  cultures  do  not  is  evidence  that  this  function  is  or 
has  been  a  variable  one.  There  is,  however,  no  evidence  to  show 
that  the  tendency  toward  variation  in  fermentation  is  any  greater 
than  in  any  other  character  used  as  a  basis  of  classification.  The 
greater  difficulty  comes  in  the  interpretation  of  the  results.  The 
objection  that  the  number  of  varieties  obtained  is  limited  only  by 
the  number  of  test  substances  used  is  valid  only  when  an  absolute 
separation  is  made  on  each  individual  reaction.  The  usual  botanical 
scheme  of  dichotomous  separation  when  applied  to  the  classification  of 
bacteria  on  the  basis  of  fermentation  tests  leads  only  to  confusion 
and  the  rejection  of  the  system.  In  the  card  arranged  for  the  classi- 
fication of  bacteria  by  a  committee  of  the  Society  of  American 
Bacteriologists,  dextrose,  saccharose,  lactose,  starch,  and  glycerin 
are  used  as  test  substances,  and  cultures  are  separated  in  the  usual 
way  on  the  fermentation  of  or  failure  to  ferment  any  one  substance. 
By  the  use  of  these  test  substances  and  similar  methods  we  could 
separate  our  nonliquefying  cultures  into  five  varieties.  But  if  it  is 
proper  to  separate  cultures  on  the  basis  of  the  fermentation  of  dex- 
trose, saccharose,  or  lactose  we  can  use  also  raffinose  and  galactose, 
and  if  glycerin  is  allowable  mannite  can  not  be  excluded,  while  inulin 
may  be  as  useful  as  starch.  Adding  these  test  substances  and  follow- 
ing the  same  principles  of  division,  we  obtain  no  less  than  14  varieties, 
and  even  these  are  not  stable,  because  the  introduction  of  a  new  test 
substance  would  probably  subdivide  them  still  more  minutely. 

Gordon  10  was  the  first  to  make  an  extensive  use  of  the  fermentation 
tests.  These  tests  were  also  used  on  an  extensive  scale  by  Andrewes 
and  Gordon  20  and  by  Houston.21  This  work  shows  the  possibilites 
of  arranging  a  large  number  of  cultures  in  groups  around  type  sets  of 
reactions.  Not  all  of  the  cultures  in  each  group  agreed  perfectly 


22  CLASSIFYING   LACTIC-ACID  BACTERIA. 

with  the  type.  Some  failed  in  one  reaction,  while  others  possessed 
some  character  not  common  to  the  entire  group.  MacConkey,18 
using  similar  methods  in  a  study  of  gas-forming  bacteria  from  milk, 
was  able  to  separate  112  cultures  into  17  groups,  and  even  these 
groups  were  sometimes  separated  by  minor  differences  only. 

On  the  basis  of  the  individual  reactions  it  was  possible  to  separate 
these  cultures  into  64  varieties.  This  work  was  continued  and 
placed  on  more  scientific  footing  by  Andrewes  and  Horder,22  and 
especially  by  Winslow  and  Rogers,  23,24  who  applied  the  principles  of 
biometry  to  the  study  of  bacteria.  In  this  way  has  been  supplied  a 
method  of  utilizing  the  physiological  tests  in  such  a  way  that  bacteria 
may  be  collected  in  natural  groups.  In  tabulating  the  characters  of 
a  large  number  of  cultures,  frequency  of  occurrence  of  those  with 
certain  common  characters  indicates  the  type,  while  the  cultures 
varying  from  these  types  occur  in  smaller  numbers  and  form  the 
connecting  links  between  the  types.  This  represents  the  state  of 
affairs  in  nature,  while  a  description  based  by  the  ordinary  method 
on  the  characters  of  a  single  culture  may  or  may  not  agree  with  the 
type. 

In  our  fermentation  tests  we  have  followed  Winslow  in  determining 
the  acidity  rather  than  the  mere  fact  of  fermentation  or  nonfermen- 
tation.  This  is  more  exact  and  sometimes  gives  additional  informa- 
tion of  value  in  separating  cultures.  The  medium  was  made  as 
follows : 

Per  cent. 

Beef  extract 0. 4 

Peptone 1.0 

Dibasic  potassium  phosphate 5 

Test  substance 2. 0 

The  use  of  dibasic  potassium  phosphate  is  of  advantage  in  that  it 
serves  to  neutralize  the  acid  and  thus  permits  a  more  active  growth 
and  higher  acid  formation.  The  acid  phosphate  formed  evidently 
checks  the  growth  when  a  certain  concentration  is  reached.  The 
neutralization  of  culture  media  by  this  means  is  discussed  by  Hender- 
son and  Webster.25 

The  cultures  were  incubated  7  days  at  30°  C.,  with  the  exception 
of  glycerin,  which  on  account  of  the  slow  fermentation  was  held  14 
days,  and  were  titrated  while  cold  against  twentieth-normal  sodium 
hydrate  with  phenolphthalein  as  an  indicator.  The  result  of  the 
titration  is  expressed  as  per  cent  of  lactic  acid.  Gas  formation  was 
determined  by  using  an  inverted  inner  tube  in  the  dextrose  broth. 
The  results  of  the  fermentation  tests  are  given  in  detail  in  Table  1  and 
are  recapitulated  in  Tables  5  and  6.  The  results  shown  in  the  two 
latter  tables  are  given  graphically  in  figures  4  and  5,  in  which  the 
frequency  of  occurrence  of  cultures  forming  certain  arbitrary  amounts 
of  acid  is  platted. 


FERMENTATION   OF   CARBOHYDRATES. 
TABLE  5. — Fermentation  of  test  substances  by  liquefying  cultures. 


23 


Test  substance. 

Per  cent  of  lactic  acid. 

Below  0.100. 

ve  0.700. 

it 

-1 

o~ 

H 

§ 

?, 

% 

i 

1 

1 

1 

£ 

% 

8 

§ 

1 

V 

V 

V 

V 

V 

? 

V 

V 

2 

2 

2 

i 

i 

jj 

i 

8 

% 

9 

a 

a 

i 

8 

& 

< 

d 

d 

o 

0 

o 

o 

o 

d 

o 

o 

d 

d 

Dextrose: 
Number  of  cultures 
Per  cent  of  total... 
Lactose: 
Number  of  cultures 
Per  cent  of  total... 
Saccharose: 
Number  of  cultures 
Per  cent  of  total  .  .  . 
Glycerin: 
N  umber  of  cultures 
Per  cent  of  total  .  .  . 
Mannite: 
Number  of  cultures 
Percent  of  total... 
Galactose: 
Number  of  cultures 
Per  cent  of  total.  .  . 

1 

3.45 

0 
0 

21 
63.  (j 

25 

75.8 

22 
66.7 

17 
53.1 

31 

100 

4 

13.8 

12 
36.4 

1 
3.0 

2 
6.1 

1 
3.0 

4 
12.5 

17.2 

11 
33.3 

3 
9.1 

1 
3.0 

2 
6.1 

5 
15.6 

19 

41.4 

4 

12.1 

0 
0 

4 
12.1 

3 
9.1 

3 
9.4 

1 
3.45 

2 
6.0 

5 
15.1 

0 
0 

3 
&1 

2 
6.3 

0 
0 

3 
9.1 

1 
3.0 

1 
3.0 

0 
0 

0 
0 

3 
10.3 

1 
3.0 

2 
6.1 

1 

3.45 

0 
0 

1 

3.45 

0 
0 

0 
0 

1 

3.45 

0 
0 

29 
33 

33 

33 

1 
3.0 

1 
3.1 

1 
3.0 

33 

32 

Raffinose  
Number  of  cultures 
Per  cent  of  total.  .  . 

31 

Inulin: 
Number  of  cultures 
Per  cent  of  total.  .  . 

22 
95.6 

.    0 
0 

0 
0 

0 
0 

0 
0 

0 
0 

1 
4.4 

23 

TABLE  6. — Fermentation  of  test  substances  by  nonliquefying  cultures. 


Test  substance. 

Per  cent  of  lactic  acid. 

Below  0.100. 

9 

? 

1 
? 

8 

t— 

?' 

Above  0.700. 

"3 

0     . 
m 

!l 

EH 

116 

117 

« 

?, 

i 

1 

I 

§ 

8 

•**• 

8 

j 

J 

j 

2 

2 

? 

? 

2 

i 

i 

8 

$ 

i 

9 

S 

8 

i 

X 

o 

o 

o 

o 

d 

o 

d 

o 

o 

o 

Dextrose: 
Number  of  cultures 
Per  cent  of  total.  .  . 
Lactose: 
Number  of  cultures 
Per  cent  of  total.  .  . 
Saccharose: 
Number  of  cultures 
Percent  of  total... 
Glycerin: 
Number  of  cultures 
Per  cent  of  total.  .  . 
Mannite: 
N  umber  of  cultures 
Per  cent  of  total.  .  . 
Galactose: 
Number  of  cultures 
Percent  of  total... 
Raffinose: 
Number  of  cultures 
Per  cent  of  total..  . 
Inulin: 
Number  of  cultures 
Percent  of  total.  .. 

1 
0.9 

3 
2.6 

75 
64.1 

93 

78.2 

78 
67.2 

8 
6.8 

90 

78.9 

101 
86.3 

0 
0 

5 
4.3 

0 
0 

2 
1.7 

I 
0.9 

5 
4.2 

0 
'  0 

0 
0 

2 
1.7 

5 
4.3 

0 
0 

12 
16.8 

0 
0 

8 
6.8 

1 
0.9 

0 
0 

4 
3.4 

8 
6.8 

1 
0.8 

7 
5.9 

3 

2.6 

19 
16.1 

1 
0.9 

2 
1.7 

10 
8.6 

16 
13.7 

2 
1.7 

4 
3.4 

4 
3.5 

28 
23.7 

0 
0 

9 

7.7 

12 
10.3 

17 
14.5 

I 
0.8 

1 
0.8 

4 
3.5 

20 
16.9 

0 
0 

5 
4.3 

30 
25.9 

30 
25.6 

3 
2.6 

13 
11.2 

31 
26.5 

8 
6.8 

10 
8.6 

1 
0.8 

7 
5.9 

12 
10.3 

0 
0 

15 
12.8 

11 

9.5 

0 
0 

3 
2.6 

4.2 

1 

0.8 

1 

0.8 

2.f 

2.e 

1 

0.8 



117 

119 

3 

2.6 

16 

13.  6 

0 
0 

0 

0 

0 

11 

9.3 

1 
0.9 

1 
0.9 

1 
0.8 

1 
0.9 

11 
9.5 

1 
0.8 

0 
0 

11 
9.5 

1 
0.8 

1 
0.9 

116 

118 

12 
10.5 

7 
6.1 

1 
0.9 

114 
117 

We  have  reckoned  any  acidity  of  below  0.1  per  cent  as  no 
fermentation,  although  this  may  be  an  arbitrary  distinction. 

In  the  work  by  Winslow  previously  cited  the  frequency  curves 
usually  showed  three  modes.  In  his  work,  however,  a  larger  num- 
ber of  cultures  selected  from  various  sources  was  used,  while  ours 


24 


CLASSIFYING   LACTIC-ACID  BACTERIA. 


came  from  milk  only.  Our  curves  usually  show  only  two  modes, 
one  at  the  point  of  no  fermentation  and  one  at  a  point  of  acidity 
varying  with  the  different  test  substances.  There  are,  however, 
certain  differences  in  the  curves  of  the  liquefiers  and  the  nonliquefiers. 
The  dextrose  curve  for  the  liquefiers  shows  that  a  large  proportion 
form  0.2  to  0.25  per  cent  of  acid,  with  a  smaller  number  at  0.35  to 


Jisy- — ~^ST- — ~^5T-    ~^T-      ~ 
./oa      ,,so      too     -zso    -jwe     •*>      *<>°     -45°     3we>    -sso 

FIG.  4.— Frequency  curves  for  acid  formation  by  the  liquefying  cultures. 

0.4,  while  with  the  nonliquefiers  there  is  a  high  point  at  0.35  to  0.4 
per  cent  and  possibly  a  second  mode  at  0.5  to  0.6. 

With  the  liquefiers  the  other  test  substances  show  two  modes  only. 
Raffinose  was  not  fermented  at  all,  and  only  one  culture  formed  acid 
from  inulin. 

The  number  of  liquefying  cultures  was  too  small  to  make  many 
deductions  therefrom,  but  it  is  easy  to  separate  these  cultures  into 


FERMENTATION   OF   CARBOHYDRATES. 


25 


two  distinct  groups.  One  of  these  forms  a  small  amount  (0.1  to  0.3 
per  cent)  of  acid  from  dextrose,  and  is  a  micrococcus  usually  appear- 
ing in  tetrads.  The  effect  on  milk  is  weak,  and  the  curdling,  which 
is  slow,  is  probably  due  more  to  the  action  of  a  rennet  than  to  the 
production  of  acid.  This  group,  as  represented  by  these  cultures,  is 
undoubtedly  heterogeneous,  and  by  the  application  of  these  methods 


80 


FIG.  5.— Frequency  curves  for  acid  formation  by  the  nonliquefylng  cultures. 

to  a  larger  number  of  cultures  would  be  split  up  in  distinct  sub- 
groups. The  second  group  of  liquefiers  is  interesting  in  that  it 
evidently  is  a  variation  from  the  typical  nonliquefying  lactic-acid 
organism.  In  its  morphology  it  is  identical  with  the  ordinary  type, 
but  differs  from  it  not  only  in  the  liquefaction  of  gelatin,  but  also 
in  usually  fermenting  glycerin.  Its  action  on  milk  is  characteristic. 


26  CLASSIFYING   LACTIC-ACID  BACTERIA. 

The  milk  is  curdled  promptly  with  a  firm  acid  curd;  digestion  begins 
at  once  and  almost  always  causes  a  separation  of  curd  from  the 
whey  down  the  side  of  the  tube. 

In  the  nonliquefying  group  there  are  with  all  the  test  substances 
only  two  distinct  modes  in  the  curves  with  the  possible  exception  of 
mannite.  In  this  case  there  are  three  modes,  one  below  0.1  per  cent* 
one  between  0.1  and  0.35,  and  another  between  0.45  and  0.5. 

Reference  to  Table  1  shows  that  of  the  8  cultures  belonging  to  the 

group  falling  between  0.1  and  0.35  per  cent,  2  were  gas  formers  and  1 

made  milk  slimy,  while  the  other  5  apparently  did  not  differ  from  those 

,  forming  a  high  acidity  or  failing  entirely  to  ferment  this  substance. 

In  arranging  these  reactions  on  the  basis  of  their  correlations,  one 
of  the  formulas  for  the  expression  of  correlation,  as,  for  instance,  that 
of  Yule,  may  be  used,  but  for  this  particular  work  the  determination 
of  the  coefficient  of  correlation  is  not  necessary.  Even  a  casual  ex- 
amination of  Table  1  shows  that  the  nonliquefiers  may  be  separated 
into  two  groups,  ;n  one  of  which  the  fermentation  is  usually  limited 
to  dextrose,  lactose,  and  galactose,  with  an  occasional  culture  fer- 
menting saccharose  or  mannite.  A  second  group  may  be  formed  of 
cultures  in  which  the  fermentative  ability  is  distinctly  higher.  These 
groups  are  illustrated  by  figure  6,  in  which  each  culture  is  placed  in 
one  of  four  groups  and  arranged  on  the  positive  or  negative  side  of 
a  dividing  line,  as  the  case  may  be,  in  each  of  the  salient  characters. 

The  division  of  the  nonliquefiers  is  not  an  arbitrary  one,  as  the 
distinction  between  the  two  groups  is  marked.  Not  only  is  there  a 
general  lack  of  fermentative  ability  in  Group  A,  but  there  is  no  cor- 
relation in  the  few  cases  of  fermentation  of  the  more  difficultly  fer- 
mentable substances.  The  fermentation  of  saccharose  is  no  indica- 
tion that  the  culture  will  ferment  mannite.  On  the  other  hand,  in 
Group  B  more  of  the  test  substances  are  fermented  and  there  is  a 
high  correlation  between  certain  activities.  The  fermentation  of 
raffinose  is  usually  correlated  with  the  fermentation  of  saccharose, 
mannite,  and  glycerin,  and  to  a  lesser  degree  with  inulin.  The  high 
fermentation  is  also  correlated  with  the  reduction  of  neutral  red. 

In  making  up  these  groups  it  was  found  that  6  cultures  (6fl,  7aa, 
7ak,  7cq,  7dw,  and  13u)  did  not  belong  in  this  collection,  and  they 
were  not  included  in  the  table.  It  will  also  be  noticed  that  7ci,  7cg, 
and  7dm  are  in  a  transition  stage  between  Groups  A  and  B,  either 
through  the  loss  of  properties  formerly  possessed  or  the  acquisition 
of  new  ones.  In  morphology  and  general  culture  characters  all  of 
the  numbers  of  this  group  agree  with  the  typical  lactic  culture.  It 
should  be  stated  that  we  obtained  all  of  the  cultures  of  Group  B  from 
one  locality,  Albert  Lea,  Minn.,  although  not  from  one  sample. 
However,  in  the  course  of  another  investigation  a  large  number  of 
cultures  which  could  be  properly  placed  in  this  group  have  been 
isolated  from  Washington  milk,  indicating  that  it  is  widely  distrib- 


GROUPING  OF   CULTURES. 


27 


tfl 


I     *  I 

FT>  -s-  -  J 


<r*. 

liJ 


Tf) 


Fm 


~_  . 

1 

I 

4? 

Ui         i 

gl 

1 

1 

<*> 

•—  ^ 

^HBB 

' 

1 

^ 

^ 

! 

1 

I* 

i 

~— 

| 

S 

3 

a  + 

<& 

J 

*o  ^ 

i 

i 
I 

GELATIN  | 

1 

LACTOSE  \ 

I 

^ 

I 

1 

1 

as 

K 

&. 

^ 

^? 

1 



\_ 

1 

I 

§^  +P^J 

V)rv  U2-J     l_l 


. 


28  CLASSIFYING    LACTIC-ACID   BACTERIA. 

uted,  although  it  does  not  occur  in  so  large  numbers  as  the  Group  A 
type.  While  the  number  of  cultures  included  in  Group  C  is  too 
small  to  permit  many  positive  deductions,  it  is  evident  that  it  rep- 
resents a  type  quite  distinct  from  A  and  B,  from  which  it  is  differ- 
entiated not  only  by  the  liquefaction  of  gelatin  but  also  by  the  cor- 
related functions  of  the  fermentation  of  mannite  and  glycerin  and 
the  failure  to  ferment  raffmose  and  inulin.  These  9  cultures  include 
3  differing  somewhat  from  the  others  morphologically,  .and  it  is 
probable  that  a  larger  collection  would  allow  a  deeper  and  more  posi- 
tive separation.  Group  D  is  made  up  of  cultures  which,  while  they 
are  of  common  occurrence  in  milk,  have  such  a  low  fermentative  ability 
that  they  probably  take  little  part  in  the  normal  souring  of  milk. 

CONCLUSIONS. 

The  stability  of  the  fermentation  tests  is  made  evident  not  only  by 
the  constancy  of  the  reactions  on  repeated  tests,  but  also  by  the 
marked  correlation  between  different  fermentative  activities  and 
between  the  fermentations  and  other  characters. 

The  usefulness  of  these  tests  is  only  apparent  when  by  means  of 
biometrical  methods  the  correlations  are  established  and  the  cultures 
are  arranged  in  groups  possessing  certain  characters  in  common,  but 
in  which  minor  variations  from  the  type  are  not  excluded. 

The  test  substances  used  can  not  be  determined  arbitrarily.  It  is 
probable  that  it  will  be  desirable  to  vary  the  test  substances  used  with 
different  groups  of  bacteria.  We  have  found  raffinose  and  glycerin  and 
the  gelatin  test  especially  valuable,  while  saccharose,  which  has  long 
been  used  for  differential  tests,  has  much  less  value.  All  of  the  groups 
have  many  cultures  fermenting  this  sugar,  and  there  is  little  correla- 
tion with  other  reactions.  While  the  determination  of  the  fermen- 
tation of  raffinose  or  glycerin  gives  one  a  good  idea  of  the  group  in 
which  the  culture  should  be  placed,  the  knowledge  that  a  culture  fer- 
ments or  fails  to  ferment  saccharose  is  of  little  assistance. 

It  should  be  remembered  that  these  cultures  were  all  selected  on 
the  basis  of  the  possession  of  a  single  positive  character,  the  fermen- 
tation of  lactose.  If  the  collection  had  been  made  on  a  broader  basis, 
it  is  highly  probable  that  the  cultures  would  have  formed  other 
groups  around  types  distinct  from  those  we  have  found  but  related 
to  them  by  certain  common  characters  and  by  transition  forms. 

The  results  recorded  in  this  paper  are  too  meager  to  warrant  any 
attempt  at  filing  names  or  establishing  the  place  of  the  lactic-acid 
bacteria  in  the  bacteriological  system,  but  we  believe  that  this  work 
indicates  that  future  efforts  in  the  direction  of  systematic  bacteriology 
should  be  toward  the  determination  of  those  characters  that  are  sig- 
nificant and  enduring  rather  than  in  fruitless  controversy  over  the 
priority  or  stability  of  some  name  based  on  descriptions  so  undeter- 
minative  that  they  convey  no  meaning. 


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30  CLASSIFYING   LACTIC-ACID  BACTERIA. 

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